Having completed the cDNA cloning of the mRNA encoding the myosin heavy chain (MHC) A isoform in chicken intestinal epithelial cells, we are presently attempting to clone the cDNA encoding the MHC-B isoform. To accomplish this, we have been screening a chicken cerebellar library and have isolated a number of clones, including one that contains approximately 230 nucleotides of 5' untranslated mRNA. Our goal is to understand if the two MHCs are functionally different and whether their expression is regulated at the level of translation, transcription, or both. We used our knowledge of the amino acid sequence of chicken intestinal epithelial cell MHC to locate the single serine residue phosphorylated in the 196 kDa MHC. The serine residue is located toward the end of the myosin rod, at residue 1915 out of 1959 residues. This serine is present in all vertebrate nonmuscle MHCs sequenced to date and evidence for its phosphorylation by protein kinase C has been found using myosin from human platelets, chicken intestinal epithelial cells and RBL-2H3 cells. Although many of the amino acids in this area are conserved in the smooth muscle MHC, the easily identifiable serine residue position is occupied by an alanine residue in the smooth muscle MHC. This explains our inability to phosphorylate the smooth muscle MHC of aortic cells with protein kinase C and suggests that there is at least one mechanism by which the activity of nonmuscle MHC may be regulated (reversible phosphorylation by protein kinase C), that is not available to smooth muscle MHCS.